Protein and Peptide Letters - Volume 31, Issue 3, 2024

Background: Heat-labile uracil-DNA glycosylase (HL-UDG) is commonly employed to eliminate carry-over contamination in DNA amplifications. However, the prevailing HL-UDG is markedly inactivated at 50°C, rendering it unsuitable for specific one-step RT-qPCR protocols utilizing reverse transcriptase at an optimal temperature of 42°C. Objective: This study aimed to explore novel HL-UDG with lower inactivation temperature and for recombinant expression. Methods: The gene encoding an HL-UDG was cloned from the cold-water fish rainbow trout (Oncorhynchus mykiss) and expressed in Escherichia coli with high yield. The thermostability of this enzyme and other enzymatic characteristics were thoroughly examined. The novel HL-UDG was then applied for controlling carry-over contamination in one-step RT-qPCR. Results: This recombinantly expressed truncated HL-UDG of rainbow trout (OmUDG) exhibited high amino acids similarity (84.1% identity) to recombinant Atlantic cod UDG (rcUDG) and was easily denatured at 40°C. The optimal pH of OmUDG was 8.0, and the optimal concentrations of both Na⁺ and K⁺ were 10 mM. Since its inactivation temperature was lower than that of rcUDG, the OmUDG could be used to eliminate carry-over contamination in one-step RT-qPCR with moderate reverse transcription temperature. Conclusion: We successfully identified and recombinantly expressed a novel HL-UDG with an inactivation temperature of 40°C. It is suitable for eliminating carry-over contamination in one-step RT-qPCR.

Background: Inhibitors of interleukin 6 [IL-6] have been utilized to treat severe COVID-19 disease. Their immunosuppressive or immunomodulating impact may be beneficial in COVID-19. Objectives: To discuss the role of IL-6 inhibitors and assess various trials conducted to evaluate the efficacy of IL-6 inhibitors in COVID-19 disease. Summary: Two of the most common causes of mortality in COVID-19-infected critically ill individuals are acute respiratory distress syndrome (ARDS) and multiorgan failure. Increased levels of inflammatory cytokines suggest that a cytokine storm, also known as cytokine release syndrome (CRS), is involved in the etiology of COVID-19. Most tissue damage, sepsis, and pulmonary and cardiovascular problems are caused mainly by the host defense system. Therefore, regulating this inflammatory cascade using immunomodulators is a prudent strategy. Although corticosteroids, as immunomodulators, are routinely used in COVID-19 management, interleukin (IL) inhibitors, especially IL-6 inhibitors, are also tested in many trials. Many studies have demonstrated that IL-6 inhibitors improve disease outcomes and decrease mortality, whereas others have shown that they are ineffective. In this paper, we briefly examined the role of IL-6 in COVID-19 pathogenesis and trials that support or refute the use of IL-6 inhibitors in treating COVID-19 disease. Results: Though mixed results are coming from trials regarding the adjuvant use of IL-6 inhibitors and standard anti-viral therapy with dexamethasone, a consensus favors using IL-6 inhibitors in severely ill COVID-19 patients regardless of the outcome.

Human blood is a window of physiology and disease. Examination of biomarkers in blood is a common clinical procedure, which can be informative in diagnosis and prognosis of diseases, and in evaluating treatment effectiveness. There is still a huge demand on new blood biomarkers and assays for precision medicine nowadays, therefore plasma/serum proteomics has attracted increasing attention in recent years. How to effectively proceed with the biomarker discovery and clinical diagnostic assay development is a question raised to researchers who are interested in this area. In this review, we comprehensively introduce the background and advancement of technologies for blood proteomics, with a focus on mass spectrometry (MS). Analyzing existing blood biomarkers and newly-built diagnostic assays based on MS can shed light on developing new biomarkers and analytical methods. We summarize various protein analytes in plasma/serum which include total proteome, protein post-translational modifications, and extracellular vesicles, focusing on their corresponding sample preparation methods for MS analysis. We propose screening multiple protein analytes in the same set of blood samples in order to increase success rate for biomarker discovery. We also review the trends of MS techniques for blood tests including sample preparation automation, and further provide our perspectives on their future directions.

Oral drug delivery is a prevalent and cost-effective method due to its advantages, such as increased drug absorption surface area and improved patient compliance. However, delivering proteins and peptides orally remains a challenge due to their vulnerability to degradation by digestive enzymes, stomach acids, and limited intestinal membrane permeability, resulting in poor bioavailability. The use of nanotechnology has emerged as a promising solution to enhance the bioavailability of these vital therapeutic agents. Polymeric NPs, made from natural or synthetic polymers, are commonly used. Natural polysaccharides, such as alginate, chitosan, dextran, starch, pectin, etc., have gained preference due to their biodegradability, biocompatibility, and versatility in encapsulating various drug types. Their hydrophobic-hydrophilic properties can be tailored to suit different drug molecules.

Objectives: In this study, we employed an in vitro culturing technique to investigate the impact of p53 on the modulation of growth-associated protein-43 (GAP-43) within the primary cortical neurons of rat specimens. Methods: (1) Within the first 24 hours after birth, the bilateral cortex was extracted from newborn Wistar rats and primary cortical neurons were cultured and identified. (2) The changes in the mRNA and protein expressions of GAP-43 induced by p53 in rat primary cortical neurons cultured in vitro were identified utilizing real-time polymerase chain reaction and western blot techniques. Results: (1) Lentiviral transfection of p53 within primary cortical neurons of rats elicited elevated levels of both mRNA and protein expressions of GAP-43, consequently culminating in a noteworthy augmentation of p53 expression. (2) The introduction of a p53 inhibitor in rat primary cortical neurons resulted in a reduction in both mRNA and protein expressions of GAP-43. Conclusion: Within primary rat cortical neurons, p53 has the potential to prompt an augmentation in both the transcriptional and protein expression levels of the GAP-43 protein.

Background: DDX3 is a protein with RNA helicase activity that is involved in a variety of biological processes, and it is an important protein target for the development of broad-spectrum antiviral drugs, multiple cancers and chronic inflammation. Objectives: The objective of this study is to establish a simple and efficient method to express and purify DDX3 protein in E. coli, and the recombinant DDX3 should maintain helicase activity for further tailor-made screening and biochemical function validation. Methods: DDX3 cDNA was simultaneously cloned into pET28a-TEV and pNIC28-Bsa4 vectors and transfected into E. coli BL21 (DE3) to compare one suitable prokaryotic expression system. The 6×His-tag was fused to the C-terminus of DDX3 to form a His-tagging DDX3 fusion protein for subsequent purification. Protein dissolution buffer and purification washing conditions were optimized. The His-tagged DDX3 protein would bind with the Ni-NTA agarose by chelation and collected by affinity purification. The 6×His-tag fused with N-terminal DDX3 was eliminated from DDX3 by TEV digestion. A fine purification of DDX3 was performed by gel filtration chromatography. Results: The recombinant plasmid pNIC28-DDX3, which contained a 6×His-tag and one TEV cleavage site at the N terminal of DDX3 sequence, was constructed for DDX3 prokaryotic expression and affinity purification based on considering the good solubility of the recombinant His-tagging DDX3, especially under 0.5 mM IPTG incubation at 18°C for 18 h to obtain more soluble DDX3 protein. Finally, the exogenous recombinant DDX3 protein was obtained with more than 95% purity by affinity purification on the Ni-NTA column and removal of miscellaneous through gel filtration chromatography. The finely-purified DDX3 still retained its ATPase activity. Conclusion: A prokaryotic expression pNIC28-DDX3 system is constructed for efficient expression and affinity purification of bioactive DDX3 protein in E. coli BL21(DE3), which provides an important high-throughput screening and validation of drugs targeting DDX3.

Background: Fungal infections in plants, animals, and humans are widespread across the world. Limited classes of antifungal drugs to treat fungal infections and loss of drug efficacy due to rapidly evolving fungal strains pose a challenge in the agriculture and health sectors. Hence, the search for a new class of antifungal agents is imperative. Cyclotides are cyclic plant peptides with multiple bioactivities, including antifungal activity. They have six conserved cysteine residues forming three disulfide linkages (C^I-C^IV, C^II-C^V, C^III-C^VI) that establish a Cyclic Cystine Knot (CCK) structure, making them extremely resistant to chemical, enzymatic, and thermal attacks. Aim: This in silico analysis of natural, plant-derived cyclotides aimed to assess the parameters that can assist and hasten the process of selecting the cyclotides with potent antifungal activity and prioritize them for in vivo/ in vitro experiments. Objective: The objective of this study was to conduct in silico studies to compare the physicochemical parameters, sequence diversity, surface structures, and membrane-cyclotide interactions of experimentally screened (from literature survey) potent (MIC ≤ 20 μM) and non-potent (MIC > 20 μM) cyclotides for antifungal activity. Methodology: Cyclotide sequences assessed for antifungal activity were retrieved from the database (Cybase). Various online and offline tools were used for sequence-based studies, such as physicochemical parameters, sequence diversity, and neighbor-joining trees. Structure-based studies involving surface structure analysis and membrane-cyclotide interaction were also carried out. All investigations were conducted in silico. Results: Physicochemical parameter values, viz. isoelectric point, net charge, and the number of basic amino acids, were significantly higher in potent cyclotides compared to non-potent cyclotides. The surface structure of potent cyclotides showed a larger hydrophobic patch with a higher number of hydrophobic amino acids. Furthermore, the membrane-cyclotide interaction studies of potent cyclotides revealed lower transfer free energy (ΔG transfer) and higher penetration depth into fungal membranes, indicating higher binding stability and membrane-disruption ability. Conclusion: These in silico studies can be applied for rapidly identifying putatively potent antifungal cyclotides for in vivo and in vitro experiments, which will ultimately be relevant in the agriculture and pharmaceutical sectors.